RU2724536C2 - Fermented milk mixture with indigestible oligosaccharides - Google Patents

Abstract

FIELD: food industry.SUBSTANCE: invention discloses application of a fermented ingredient and indigestible oligosaccharide to increase IgA secretion in infants. Also disclosed is a method of increasing IgA secretion in infants.EFFECT: this group of inventions ensures production of a product suitable for infants ration.7 cl, 2 tbl, 2 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to the nutrition of infants and young children to enhance the immune system.

BACKGROUND OF THE INVENTION

It is generally recognized that the best nutrition for a newborn is breast milk. When a mother cannot breastfeed her baby or does not want to breastfeed, infant formula (IF) (first-level infant formula) based on mature breast milk is considered the best alternative. A study aimed at improving the quality of the infant formula does not necessarily aim to reproduce the exact composition of breast milk, but is aimed at achieving functional effects in addition to just the nutritional aspects that are observed in breast-fed infants.

Breast milk is rich in secretory IgA (sIgA), which serves as the first line of defense in protecting the infant's intestinal barrier from intestinal toxins and pathogens. SIgA is resistant to digestive enzymes and helps to remove antigens and pathogens from the intestines, blocking their access to epithelial receptors, trapping them in mucus and facilitating their removal through peristaltic and ciliary epithelial activity. In addition, sIgA functions in the immune system to protect the mucous membrane and intestinal homeostasis, without provoking, but even reducing the inflammatory response. In contrast, sIgA concentrations are significantly lower in standard infant formula based on cow's milk ingredients, and infants fed standard infant formula have lower sIgA levels compared to breastfed babies.

EP 2318046 describes the combination of probiotics with secretory IgA to treat or prevent inflammation. EP 2315595 describes the use of live probiotic bacteria to increase the secretion of immunoglobulin in infants after cesarean section. However, this document does not describe a fermented ingredient or lactic acid. WO 2014/201037 describes the use of probiotics, prebiotics, synbiotics and antibiotics for treating microbiotic changes in mammals associated with antibiotic treatment and various immunological conditions. Fukushima et al. (1998, Int J Food Micribiol 42: 39-44) studied the effect of a probiotic strain of Bifidobacterium lactis on IgA secretion in a cohort of older children and found elevated levels of total IgA and IgA against poliovirus. Probiotics are live lactic acid bacteria, such as live lactobacilli or live bifidobacteria. Typically, a baby formula with probiotics is sold in the form of powders with dried probiotic cells in which fermentation is not possible due to unfavorable conditions for fermentation, for example, too low water activity, and no lactic acid is formed.

Non-digestible (non-digestible) oligosaccharides affect the production of sIgA in the intestine through their effect on the composition of the microbiota. Scholtens et al. (2008, J Nutr 138: 1141-1147), as well as Bakker-Zierikzee et al. (2006, Pediatr Allergy Immunol 17: 134-140), found higher sIgA levels in the feces of healthy babies fed milk a mixture supplemented with scGOS / lcFOS than in infants fed standard milk formula, thereby confirming this hypothesis.

Mullie et al. (2004, Ped Res 56: 791-795) found that the intake of fermented infant formula in healthy newborns was accompanied by increased production of poliovirus-specific sIgA antibodies in the intestine after vaccine administration, but not with effect on total IgA titers.

WO 2009/151330 describes a composition obtained by fermentation by lactic acid bacteria and indigestible oligosaccharides for use in children after cesarean section. The goal is to challenge tolerance to support subsequent colonization with living bacteria. IgA secretion is not mentioned. WO 2009/151331 describes an immunostimulatory composition comprising indigestible oligosaccharides and a product obtained after incubation with bifidobacteria. By stimulating the Th1 response and decreasing the Th2 response of the systemic immune system, the response to vaccination increases and allergies decrease. WO 2009/151329 describes a composition comprising indigestible oligosaccharides and a product obtained by incubation with bifidobacteria to reduce bacterial translocation and improve intestinal barrier function. The purpose of this is to prevent systemic infections in this way. The document does not mention any effect on the immune defense of the mucous membrane and the secretion of IgA.

SUMMARY OF THE INVENTION

In clinical trials with healthy, full-term newborns, it was found that the administration of a fermented infant formula containing indigestible oligosaccharides showed a significant increase in intestinal secretory IgA (sIgA) compared to the administration of an unfermented control infant formula without indigestible oligosaccharides, a fermented infant formula without indigestible or oligosaccharides unfermented milk mixture containing indigestible oligosaccharides. The secretion of sIgA was synergistically increased and was more reminiscent of sIgA in the control group of breastfeeding. This also indicates a synergistic strengthening of the immune system to protect the mucous membrane.

Surprisingly, the synergistic effect cannot be explained by the direct interaction between the indigestible oligosaccharides and lactic acid bacteria in the fermented composition, since the bacteria were inactivated, or the increased activity of microorganisms in the intestine, since there was no clear correlation with the pH in the intestine.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Thus, the present invention relates to a method for increasing IgA secretion in a human subject from 0 to 36 months of age, comprising administering to the human subject a food composition containing a fermented ingredient and a non-digestible oligosaccharide, wherein said food composition contains from 0.02 to 1, 5 weight % of the amount of lactic acid and lactate calculated on the dry weight of the food composition and contains from 2.5 to 15 weight. % indigestible oligosaccharide based on the dry weight of the food composition.

In one embodiment, the method of increasing IgA secretion in a human subject aged 0 to 36 months of the present invention is a non-therapeutic method.

The present invention can also be formulated as the use of a fermented ingredient and a non-digestible oligosaccharide in the manufacture of a food composition for use in increasing IgA secretion in a human subject aged 0 to 36 months, the food composition comprising from 0.02 to 1.5 weight. % of the sum of lactic acid and lactate based on the dry weight of the food composition and contains from 2.5 to 15% by weight of indigestible oligosaccharide based on the dry weight of the food composition.

The present invention can also be formulated as a food composition containing a fermented ingredient and a non-digestible oligosaccharide, wherein the food composition contains from 0.02 to 1.5 weight. % of the amount of lactic acid and lactate calculated on the dry weight of the food composition and contains from 2.5 to 15 weight. % indigestible oligosaccharide based on the dry weight of the food composition, for use in increasing IgA secretion in a human subject aged 0 to 36 months.

In another aspect, the present invention relates to a method for enhancing the immune defense of a mucous membrane in a human subject aged 0 to 36 months, comprising administering to the human subject a food composition comprising a fermented ingredient and an indigestible oligosaccharide, said food composition comprising from 0.02 to 1.5 weight % of the amount of lactic acid and lactate calculated on the dry weight of the food composition and contains from 2.5 to 15 weight. % indigestible oligosaccharide based on the dry weight of the food composition.

In one embodiment, a method of enhancing the immune defense of a mucous membrane in a human subject aged 0 to 36 months of the present invention is a non-therapeutic method.

The present invention can also be formulated as the use of a fermented ingredient and a non-digestible oligosaccharide in the manufacture of a food composition for use in enhancing the immune defense of the mucous membrane of a human subject aged 0 to 36 months, the food composition comprising from 0.02 to 1.5 weight. % of the amount of lactic acid and lactate calculated on the dry weight of the food composition and contains from 2.5 to 15 weight. % indigestible oligosaccharide based on the dry weight of the food composition.

The present invention can also be formulated as a food composition containing a fermented ingredient and a non-digestible oligosaccharide, wherein the food composition contains from 0.02 to 1.5 weight. % of the amount of lactic acid and lactate calculated on the dry weight of the food composition and contains from 2.5 to 15 weight. % indigestible oligosaccharide based on the dry weight of the food composition, for use in enhancing the immune defense of the mucous membrane of a human subject aged 0 to 36 months.

Fermented ingredient

The food composition in the methods or applications of the present invention, hereinafter also referred to as the food composition of the present invention, contains a fermented ingredient. A fermented ingredient is a composition that is fermented with lactic acid bacteria. Fermentation preferably occurs during the manufacturing process of the food composition. Preferably, the food composition does not contain significant amounts of viable bacteria in the final product due to inactivation by heat treatment after fermentation or inactivation by other methods. Preferably, the fermented ingredient is a product of milk origin, which is a milk substrate, which is fermented by lactic acid bacteria, and said milk substrate includes at least one selected from the group consisting of milk, whey, whey protein, whey protein hydrolyzate, casein, casein hydrolyzate and lactose, or mixtures thereof. Accordingly, food compositions containing a fermented ingredient and a non-digestible oligosaccharide, and a method for their preparation are described in WO 2009/151330, WO 2009/151331 and WO 2013/187764.

The fermented ingredient preferably includes fragments of bacterial cells such as glycoproteins, glycolipids, peptidoglycan, lipoteichoic acid (LTA), lipoproteins, nucleotides and / or capsule polysaccharides. It is advisable to use a fermented ingredient, including inactivated bacteria and / or cell fragments directly entering the final food product, as this will lead to a higher concentration of bacterial cell fragments. When commercial preparations are used, they are usually washed and the material is separated from the aqueous growth medium containing bacterial cell fragments, thereby reducing or eliminating the presence of bacterial cell fragments. In addition, after fermentation and / or other interactions of lactic acid bacteria with a milk substrate, additional biologically active compounds are formed, such as bioactive peptides and / or oligosaccharides, which also stimulate the immune system to protect the mucous membrane. Therefore, a fermented ingredient, in particular a fermented product of dairy origin, is believed to have an increased effect compared to an unfermented product of dairy origin.

Preferably, when the food composition contains from 5 to 97.5 weight. % fermented ingredient based on dry weight, more preferably from 10 to 95 weight. %, more preferably from 20 to 90 weight. %, even more preferably from 25 to 60 weight. % As a method for determining the degree of fermentation, the level of the sum of lactic acid and lactate can be measured in the food composition, since this is the metabolic end product formed by lactic acid bacteria during fermentation. The food composition of the present invention contains from 0.02 to 1.5 weight. % of the sum of lactic acid and lactate based on the dry weight of the composition, more preferably from 0.05 to 1.0 weight. %, even more preferably from 0.05 to 0.5 weight. % Preferably, at least 50 weight. %, even more preferably at least 90 weight. % of the sum of lactic acid and lactate is in the form of L (+) - isomer. Thus, in one embodiment, the sum of L (+) - lactic acid and L (+) - lactate is more than 50 weight. %, more preferably more than 90 weight. %, calculated on the sum of the total lactic acid and lactate. Here, L (+) - lactate and L (+) - lactic acid are also referred to as L-lactate and L-lactic acid.

Lactic acid bacteria used to produce the fermented ingredient.

Lactic acid bacteria used to prepare the fermented ingredient, in particular for the fermentation of the milk substrate, are preferably provided as a mono- or mixed culture. Lactic acid bacteria are composed of the genera Bifidobacterium, Lactobacillus, Carnobacterium, Enterococcus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. Preferably, the lactic acid bacteria used for fermentation include bacteria of the genus Bifidobacterium and / or Streptococcus.

Preferably, Streptococcus is a S. thermophilic strain. Preferably, Streptococcus exhibits beta-galactosidase activity during substrate fermentation. The selection of a suitable strain of S. thermophilus is described in Example 2 of EP 778885 and in Example 1 of FR 2723960. In yet another preferred embodiment, in accordance with the present invention, the food composition comprises 10 ² -10 ⁵ CFU of living S bacteria . thermophilus, per g dry weight of the food composition, food composition preferably comprises 10 ² to 10 ⁴ cfu of live bacteria S. thermophilus per g dry weight.

Preferred strains of S. thermophilus for the preparation of the fermented ingredient for the purpose of the present invention were deposited by Compagnie Gervais Danone at the Collection Nationale de Cultures de Microorganismes (CNCM) at the initiative of the Pasteur Institute, 25 rue du Docteur Roux, Paris, France, August 23, 1995 under inventory number I-1620 and August 25, 1994 under inventory number I-1470.

Bifidobacteria are gram-positive, anaerobic, rod-shaped bacteria. Preferred Bifidobacterium species for preparing the fermented ingredient for the purpose of the present invention are preferably at least 95% identical in sequence to the 16S rRNA of the type strain of the corresponding Bifidobacterium species, more preferably at least 97% identical, as defined in reference books on this subject, for example, Sambrook , J., Fritsch, EF and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor (NY) Laboratory Press. Bifidobacteria, which are preferably used, are also described by Scardovi, V. Genus Bifidobacterium. p.1418-p.1434. In: Bergey's manual of systematic Bacteriology. Vol. 2. Sneath, PHA, NS Mair, ME Sharpe and JG Holt (ed.). Baltimore: Williams & Wilkins. 1986. 635 p. Preferably, the lactic acid bacteria used for fermentation include or represent at least one Bifidobacterium species selected from the group consisting of B. breve, B. infantis, B. bifidum, B. catenulatum, B. adolescentis, B. thermophilum , B. gallicum, B. animalis or lactis, B. angulatum, B. pseudocatenulatum, B. thermacidophilum, and B. longum, more preferably B. breve, B. infantis, B. bifidum, B. catenulatum, B. longum , more preferably B. longum and B. breve , even more preferably B. breve , more preferably B. breve , selected from the group consisting of B. breve Bb-03 (Rhodia / Danisco), B. breve M-16V (Morinaga), B. breve R0070 (Institute Rosell, Lallemand), B. breve BR03 (Probiotical), B. breve BR92 (Cell Biotech) DSM 20091, LMG 11613 and B. breve I-2219, which are deposited in CNCM, Paris, France. Most preferably, B. breve is B. breve M-16V (Morinaga) or B. breve I-2219, even more preferably B. breve I-2219.

Most preferably, the food composition contains a fermented ingredient that is fermented with lactic acid bacteria, including both B. breve and S. thermophilus . In one embodiment, the lactic acid bacteria fermentation is the fermentation of Streptococcus thermophilus and Bifidobacterium breve . In one embodiment, the food composition comprises a fermented ingredient, wherein the lactic acid bacteria are inactivated after fermentation.

Preferably, the fermented ingredient is not fermented with Lactobacillus bulgaricus . Fermented products using L. bulgaricus are considered unsuitable for infants, since in infants in the first months of life, specific dehydrogenase, which converts D-lactate to pyruvate, is much less active than dehydrogenase, which converts L-lactate.

Preferably, the food composition of the present invention contains inactivated lactic acid and / or bacterial fragments derived from lactic acid bacteria obtained from more than 1 × 10 ¹⁴ CFU of milk bacteria per gram based on the dry weight of the final composition, more preferably 1 × 10 ⁵ CFU, even more preferably 1 × 10 ⁶ CFU. Preferably, inactivated bacteria or bacterial fragments are obtained from less than 1 × 10 ¹⁴ CFU of milk bacteria per gram based on the dry weight of the final composition, more preferably 1 × 10 ¹³ CFU, even more preferably 1 × 10 ¹² CFU.

Fermentation process

The milk substrate to be fermented is suitably present in the aqueous medium. The milk substrate to be fermented is preferably selected from the group consisting of milk, whey, whey protein, whey protein hydrolyzate, casein, casein hydrolyzate and lactose, and mixtures thereof. The milk may be whole milk, semi-skim milk and / or skim milk. Preferably, the milk substrate to be fermented includes skim milk. The whey may be sweet whey and / or acid whey. Preferably, whey is present in a concentration of from 3 to 80 g dry weight per liter of aqueous medium containing a milk substrate, more preferably 40 to 60 g per liter. Preferably, the whey protein hydrolyzate is present in an amount of 2 to 80 g dry weight per liter of aqueous medium containing a milk substrate, more preferably 5 to 15 g / L. Preferably, lactose is present in an amount of 5-50 g dry weight per liter of aqueous substrate, more preferably 1 to 30 g / l. Preferably, the aqueous medium containing the milk substrate contains buffer salts to maintain the pH in the desired range. Sodium or potassium phosphate monosubstituted, preferably in an amount of from 0.5 to 5 g / l, more preferably from 1.5 to 3 g per liter, is preferably used as the buffer salt. Preferably, the aqueous medium containing the milk substrate contains cysteine in an amount of from 0.1 to 0.5 g per liter of aqueous substrate, more preferably from 0.2 to 0.4 g / L. The presence of cysteine leads to a low redox potential of the substrate, which is beneficial for the activity of lactic acid bacteria, in particular bifidobacteria. Preferably, the aqueous medium containing the milk substrate contains the yeast extract in an amount of from 0.5 to 5 g / l of the aqueous medium containing the milk substrate, more preferably from 1.5 to 3 g / l. Yeast extract is rich in a source of cofactors of enzymes and growth factors of lactic acid bacteria. The presence of yeast extract enhances the fermentation of lactic acid bacteria.

Accordingly, when the milk substrate, in particular the aqueous medium containing the milk substrate, is pasteurized before the fermentation step, in order to exclude the presence of undesired living bacteria. Accordingly, when the product is pasteurized after fermentation to inactivate the enzymes. Accordingly, when the enzyme inactivation takes place at 75 ^ο C for 3 min. Accordingly, when the aqueous medium containing the milk substrate is homogenized before fermentation, and / or the milk product is homogenized after fermentation. Homogenization results in a more stable substrate and / or fermented product, especially in the presence of fat.

The inoculation density is preferably from 1 × 10 ² to 5 × 10 ¹⁰ , preferably from 1 × 10 ⁴ to 5 × 10 ⁹ CFU of lactic acid bacteria / ml of an aqueous medium containing a milk substrate, more preferably from 1 × 10 ⁷ to 1 × 10 ⁹ CFU of lactic acid bacteria / ml of an aqueous medium containing a milk substrate. The final bacterial density after fermentation is preferably from 1 × 10 ³ to 1 × 10 ¹⁰ , more preferably from 1 × 10 ⁴ to 1 × 10 ⁹ CFU / ml, of an aqueous medium containing a milk substrate.

The fermentation is preferably conducted at a temperature from about 20 ^{ο ο} C to 50 C, more preferably from 30 C to 45 ^{ο ο} C, more preferably from about 37 ^{ο ο} C to 42 C. The optimum temperature for growth and / or activity of lactic acid bacteria, more specifically, lactobacilli and / or bifidobacteria, ranges from 37 ^° C to 42 ^° C.

The incubation is preferably carried out at a pH of from 4 to 8, more preferably from 6 to 7.5. This pH does not cause protein precipitation and / or an undesirable taste, while lactic acid bacteria such as lactobacilli and / or bifidobacteria are capable of fermenting the milk substrate.

The incubation time is preferably from 10 minutes to 48 hours, preferably from 2 hours to 24 hours, more preferably from 4 to 12 hours. A sufficiently long time allows the passage of fermentation and the accompanying production of immunogenic fragments of cells, such as glycoproteins, glycolipids, peptidoglycan, lipoteichoic acid (LTA), flagella, lipoproteins, DNA and / or capsule polysaccharides, to a high degree, while the incubation time does not have to be unnecessarily long for economic reasons.

Preferably, the milk substrate, preferably skim milk, is pasteurized, cooled and fermented with one or more acidic acid producing strains, preferably S. thermophilus strain, to a certain degree of acidity in which the fermented product is cooled and stored. Preferably, the second milk product is obtained in a similar manner using one or more types of Bifidobacterium for fermentation. Subsequently, the two fermented products are preferably mixed together and mixed with other components that make up the baby mixture, with the exception of the fat component. Preferably, the mixture is preheated, and subsequently the fat is added in turn, homogenized, pasteurized and dried.

Procedures for preparing fermented ingredients suitable for the purpose of the present invention are known per se. In EP 778885, which is incorporated herein by reference, describes, in particular in example 7, a suitable method for producing a fermented ingredient. In FR 2723960, which is incorporated herein by reference, describes, in particular in example 6, a suitable method for producing a fermented ingredient. Briefly, a milk substrate, preferably pasteurized, containing lactose and optionally additional macronutrients such as fats, preferably vegetable fats, casein, whey protein, vitamins and / or minerals, etc., is concentrated to, for example, from 15 to 50% dry substances, and then S. thermophilus is inoculated into it, for example, 5% of a culture containing 10 ⁶ to 10 ¹⁰ bacteria per ml. Preferably, this milk substrate includes milk protein peptides. The temperature and duration of the fermentation are indicated above. Accordingly, after fermentation, the fermented ingredient can be pasteurized or sterilized and, for example, spray dried or lyophilized to obtain a form suitable for constitution in the final product.

A preferred method for producing the fermented ingredient of the present invention is described in WO 01/01785, more specifically in Examples 1 and 2. A preferred method for producing the fermented ingredient of the present invention is described in WO 2004/093899, more specifically in Example 1.

Methods of inactivation and / or physical removal of living cells

Living cells of lactic acid bacteria in the fermented ingredient after fermentation are preferably removed, for example, by inactivation and / or physical removal. Cells are preferably inactivated. Preferably, the lactic acid bacteria are destroyed by heat treatment after fermentation of the milk substrate. Preferred methods of destruction by heat treatment are (instant) pasteurization, sterilization, ultra-high temperature treatment, high temperature / short-term heat treatment and / or spray drying at temperatures at which the bacteria do not survive. Cell fragments are preferably obtained by heat treatment. In this heat treatment, preferably at least 90% of the living microorganisms are inactivated, more preferably at least 95%, even more preferably at least 99%. Preferably, the fermented food composition contains less than 1 × 10 ⁵ colony forming units (CFU) of live lactic acid bacteria per gram of dry weight. The heat treatment is preferably carried out at a temperature in the range from 70 to 180 ^° C, preferably from 80 to 150 ^° C, preferably within about 3 minutes to 2 hours, preferably in the range from 80 to 140 ^° C for 5 minutes to 40 minutes. Inactivation of lactic acid bacteria predominantly leads to less post-oxidation and a safer product. This is especially beneficial when the food composition is to be administered to infants or young children. Accordingly, after fermentation, the fermented ingredient can be pasteurized or sterilized and, for example, spray dried or lyophilized to obtain a form suitable for constitution in the final product.

Indigestible oligosaccharide

The food composition of the present invention contains a non-digestible oligosaccharide and preferably contains at least two different non-digestible oligosaccharides, in particular two different sources of non-digestible oligosaccharide. The presence of a non-digestible oligosaccharide stimulates sIgA secretion. The presence of a non-digestible oligosaccharide stimulates the immune system to protect the mucous membrane. Therefore, the presence of both a non-digestible oligosaccharide and a fermented ingredient, in particular a milk product obtained by fermentation by lactic acid bacteria, synergistically and predominantly leads to greater secretion of IgA in the intestine. Therefore, the presence of both a non-digestible oligosaccharide and a fermented ingredient, in particular a product of milk origin obtained by fermentation by lactic acid bacteria, synergistically and mainly leads to increased immune defense of the mucous membrane.

As used herein, the term “oligosaccharide” refers to saccharides with a degree of polymerization (DP) of from 2 to 250, preferably with DP from 2 to 100, more preferably from 2 to 60, even more preferably from 2 to 10. If an oligosaccharide with DP from 2 to 100 is included in the food composition of the present invention, this leads to compositions that may contain oligosaccharides with DP from 2 to 5, DP from 50 to 70 and DP from 7 to 60. As used in the present invention, the term “non-digestible oligosaccharide” refers to oligosaccharides which do not break down in the intestine by the action of acids or digestive enzymes present in the upper part of the human digestive tract, for example, the small intestine and stomach, but which are preferably fermented by the microbiota of the human intestine. For example, sucrose, lactose, maltose, and maltodextrins are considered digestible.

Preferably, the non-digestible oligosaccharide of the present invention is soluble. As used herein, the term “soluble” when referring to a polysaccharide, filament or oligosaccharide means that the substance is at least soluble in accordance with the method described by L. Prosky et al., J. Assoc. Off. Anal. Chem. 71, 1017-1023 (1988).

The non-digestible oligosaccharide included in the food compositions of the present invention in the methods or applications of the present invention preferably includes a mixture of non-digestible oligosaccharides. Non-digestible oligosaccharide is preferably selected from the group consisting of fructooligosaccharide, such as inulin, indigestible dextrins, galactooligosaccharides such as transgalactooligosaccharide, xylooligosaccharides, arabinooligosaccharides, arabinogalaktooligosaharida, glucooligosaccharides, gentiooligosaharida, glyukomannooligosaharida, galactomanno-oligosaccharides, mannan-oligosaccharides, isomaltooligosaccharides, nigerooligosaharida, glyukomannooligosaharida, chitooligosaccharide, soy oligosaccharide, uronic acid oligosaccharide, sialyl oligosaccharide such as 3-sialyllactose (3-SL), 6-sialyllactose (6-SL), lactosialyltetrasaccharide a, b, c (LST), DS-tetral ), sialyl lacto-N-hexaose (S-LNH), DS-LNH, and a fuco-oligosaccharide such as the (un) sulfated oligosaccharide fucoidan, 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL) , difucosyllactose, lacto-N-fucopenatosis, (LNFP) I, II, III, V, lacto-N-neofucopenosis (LNnFP), lacto-N-difucosylhexaose (LNDH), and their mixtures are even more preferably selected from the group consisting of a fructooligosaccharide such as inulin, a galactooligosaccharide such as a transgalactooligosaccharide, and a fucoooligosaccharide and mixtures thereof, even more preferably a transgalactooligosaccharide and / or inulin, most preferably transgalactosigaride. In one embodiment, in the composition or methods of the present invention, the non-digestible oligosaccharide is selected from the group consisting of transgalactooligosaccharide, fructooligosaccharide, and mixtures thereof.

The indigestible oligosaccharide is preferably selected from the group consisting of β-galactooligosaccharide, α-galactooligosaccharide and galactan. According to a more preferred embodiment, the non-digestible oligosaccharide is β-galactooligosaccharide. Preferably, a non-digestible oligosaccharide includes a galactooligosaccharide with β (1,4) -, β (1,3) - and / or β (1,6) -glycoside bonds and terminal glucose. A transgalactooligosaccharide, for example, is available under the trade name Vivinal®GOS (Domo FrieslandCampina Ingredients), Bi2muno (Clasado), Cup-oligo (Nissin Sugar) and Oligomate55 (Yakult). These oligosaccharides increase sIgA levels and enhance the mucosal immune defense activity to a greater extent.

The non-digestible oligosaccharide preferably includes a fructooligosaccharide. The fructooligosaccharide may in a different context have names such as fructopolysaccharide, oligofructose, polyfructose, polyfructan, inulin, levan and fructan, and may refer to oligosaccharides containing β-linked fructoses as units that are preferably linked to β (2,1) - and / or β (2,6) -glycoside bonds, and with a preferred DP of 2 to 200. Preferably, the fructooligosaccharide contains a terminal glucose linked via a β (2,1) -glycoside linkage. Preferably, the fructooligosaccharide contains at least 7 β-linked fructoses as units. In another preferred embodiment, inulin is used. Inulin is a type of fructooligosaccharide in which at least 75% of the glycosidic bonds are β (2,1) bonds. Inulin typically has an average chain length of 8 to 60 monosaccharides as units. Suitable fructooligosaccharide for use in the compositions of the present invention is commercially available under the trade name Raftiline®HP (Orafti). Other suitable sources are Raftilose (Orafti), Fibrulose and Fibrulin (Cosucra), and Frutafit and Frutalosa (Sensus).

Preferably, the food composition of the present invention comprises a mixture of galactooligosaccharide and fructooligosaccharide. Preferably, the mixture of galactooligosaccharide and fructooligosaccharide is present in a weight ratio of 1/99 to 99/1, more preferably 1/19 to 19/1, more preferably 1/1 to 19/1, more preferably 2/1 to 15/1 more preferably from 5/1 to 12/1, even more preferably from 8/1 to 10/1, even more preferably in an approximate ratio of 9/1. This weight ratio is particularly advantageous when the galactooligosaccharide has a low average DP and the fructooligosaccharide has a relatively high DP. Most preferred is a mixture of a galactooligosaccharide with an average DP below 10, preferably below 6, and a fructose oligosaccharide with an average DP above 7, preferably above 11, even more preferably above 20. Such a mixture synergistically increases sIgA levels and enhances the mucosal immune defense activity.

Preferably, the food composition of the present invention comprises a mixture of short chain fructooligosaccharide and long chain fructooligosaccharide. Preferably, the mixture of short chain fructooligosaccharide and long chain fructooligosaccharide is present in a weight ratio of 1/99 to 99/1, more preferably 1/19 to 19/1, even more preferably 1/10 to 19/1, more preferably 1/5 to 15/1, more preferably 1/1 to 10/1. A mixture of short chain fructooligosaccharide with an average DP below 10, preferably below 6, and a fructooligosaccharide with an average DP above 7, preferably above 11, even more preferably above 20, is preferred.

The food composition of the present invention contains from 2.5 to 20 weight. % of the total indigestible oligosaccharide, more preferably from 2.5 to 15 weight. %, even more preferably from 3.0 to 10 weight. %, most preferably from 5.0 to 7.5 weight. % based on the dry weight of the food composition. Per 100 ml, the food composition of the present invention preferably contains from 0.35 to 2.5 weight. % of the total indigestible oligosaccharide, more preferably from 0.35 to 2.0 weight. %, even more preferably from 0.4 to 1.5 weight. %, based on 100 ml of the food composition. A lower amount of indigestible oligosaccharide will be less effective in stimulating the formation of sIgA or in the immune defense of the mucous membrane, while too high an amount will lead to side effects of bloating and discomfort in the stomach.

Food composition

The food composition used in accordance with the present invention can also be considered as a pharmaceutical composition, preferably suitable for administration to infants. The food composition of the present invention is preferably administered enterally, more preferably orally.

The food composition of the present invention is preferably a baby formula (first level milk formula), second level milk mixture or third level milk. The food composition of the present invention can be advantageously used as a balanced diet for infants. Preferably, the food composition of the present invention is an infant formula. An infant formula is defined as an infant formula for use for infants, and may be, for example, a first-level infant formula for infants from 0 to 6 or from 0 to 4 months. The second level infant formula is intended for infants from 4 or 6 months to 12 months of age. Milk of the third level is intended for children aged 12 to 36 months. The composition of the present invention preferably contains a lipid component, a protein component and a carbohydrate component, and is preferably administered in liquid form. The food composition of the present invention may also be in the form of a dry food product, preferably in the form of a powder, which is accompanied by instructions for mixing said dry food product, preferably a powder, with a suitable liquid, preferably water. The food composition used in accordance with the present invention preferably contains other parts such as vitamins, minerals, trace elements and other trace elements to make it a balanced food composition. Preferably, infant formulas contain vitamins, minerals, trace elements and other trace elements in accordance with international directives.

The food composition of the present invention preferably contains a lipid, protein, and digestible carbohydrate, wherein the lipid provides 5 to 50% of the total calories, the protein provides 5 to 50% of the total calories, and the digestible carbohydrate provides 15 to 90% of the total calories. Preferably, in the food composition of the present invention, the lipid provides from 35 to 50% of the total calories, the protein provides from 7.5 to 12.5% of the total calories, and the digestible carbohydrate provides from 40 to 55% of the total calories. To calculate% of the total calories for protein, you need to take into account the total amount of energy released by proteins, peptides and amino acids. Preferably, the lipid provides from 3 to 7 g of lipid per 100 kcal, preferably from 4 to 6 g per 100 kcal, the protein provides from 1.6 to 4 g per 100 kcal, preferably from 1.75 to 2.5 g per 100 kcal, and the digestible carbohydrate provides from 5 to 20 g per 100 kcal, preferably from 8 to 15 g per 100 kcal of the food composition. Preferably, the food composition of the present invention comprises a lipid providing from 4 to 6 g per 100 kcal, a protein providing from 1.6 to 1.9 g per 100 kcal, more preferably from 1.75 to 1.85 g per 100 kcal, and digestible carbohydrate providing from 8 to 15 g per 100 kcal of food composition. In one embodiment, the lipid provides from 3 to 7 g of lipid per 100 kcal, preferably from 4 to 6 g per 100 kcal, the protein provides from 1.6 to 2.1 g per 100 kcal, preferably from 1.6 to 2.0 g per 100 kcal, and the digestible carbohydrate provides from 5 to 20 g per 100 kcal, preferably from 8 to 15 g per 100 kcal of the food composition, and preferably the digestible carbohydrate component contains at least 60 weight. % lactose based on total digestible carbohydrate, more preferably at least 75 weight. %, even more preferably at least 90 weight. % lactose based on total digestible carbohydrate. The amount of all calories is determined by the sum of calories obtained from protein, lipids, digestible carbohydrates, and indigestible oligosaccharide.

The food composition of the present invention preferably contains a digestible carbohydrate component. Preferred digestible carbohydrate components are lactose, glucose, sucrose, fructose, galactose, maltose, starch and maltodextrin. Lactose is the main digestible carbohydrate present in breast milk. The food composition of the present invention preferably contains lactose. Since the food composition of the present invention contains a fermented ingredient, which is obtained by fermentation by lactic acid bacteria, the amount of lactose is reduced compared to its source due to fermentation, in which lactose is converted into lactate and / or lactic acid. Therefore, in preparing the food composition of the present invention, lactose is preferably added. Preferably, the food composition of the present invention does not contain large amounts of carbohydrates other than lactose. Compared to digestible carbohydrates such as maltodextrin, sucrose, glucose, maltose and other digestible carbohydrates with a high glycemic index, lactose has a lower glycemic index and is therefore preferred. The food composition of the present invention preferably contains a digestible carbohydrate, with at least 35 weight. %, more preferably at least 50 weight. %, more preferably at least 60 weight. %, more preferably at least 75 weight. %, even more preferably at least 90 weight. %, most preferably at least 95 weight. % of digestible carbohydrate is lactose. Based on dry weight, the food composition of the present invention preferably contains at least 25 weight. % lactose, preferably at least 40 weight. %, more preferably at least 50 weight. % lactose.

The food composition of the present invention preferably contains at least one lipid selected from the group consisting of animal lipid (excluding human lipids) and plant lipids. Preferably, the food composition of the present invention contains a combination of vegetable lipids and at least one oil selected from the group consisting of fish oil, animal oil, algae oil, mushroom oil and bacterial oil. The lipid of the food composition of the present invention preferably provides from 3 to 7 g per 100 kcal of the food composition, preferably the lipid provides from 4 to 6 g per 100 kcal. When in liquid form, for example, as a ready-to-drink liquid, the food composition preferably contains from 2.1 to 6.5 g of lipid per 100 ml, more preferably from 3.0 to 4.0 g per 100 ml. Based on dry weight, the food composition of the present invention preferably contains from 12.5 to 40 weight. % lipid, more preferably from 19 to 30 weight. % Preferably, the lipid includes essential fatty acids: alpha-linolenic acid (ALA), linoleic acid (LA) and / or long chain polyunsaturated fatty acids (LC-PUFA). LC-PUFA, LA, and / or ALA may be provided as free fatty acids, in the form of triglycerides, in the form of diglycerides, in the form of monoglycerides, in the form of phospholipids, or as a mixture of one of the above. Preferably, the food composition of the present invention contains at least one, preferably at least two, lipid sources selected from the group consisting of rapeseed oil (such as rapeseed oil, low erucic acid rapeseed oil and canola oil) , high oleic acid sunflower oil, high oleic acid safflower oil, olive oil, sea oils, microbial oils, coconut oil, palm kernel oil and milk fat. The food composition of the present invention is not breast milk.

The food composition of the present invention preferably contains protein. The protein used in the food composition is preferably selected from the group consisting of animal proteins other than humans, preferably milk proteins, vegetable proteins, such as preferably soy protein and / or rice protein, and mixtures thereof. The food composition of the present invention preferably contains casein and / or whey protein, more preferably bovine whey proteins and / or bovine casein. Thus, in one embodiment, the protein in the food composition of the present invention includes a protein selected from the group consisting of whey protein and casein, preferably whey protein and casein, preferably whey protein and / or casein from cow's milk. Preferably, the protein is less than 5 weight. % based on the total protein of free amino acids, dipeptides, tripeptides or hydrolyzed protein. The food composition of the present invention preferably contains casein and whey proteins in a weight ratio of casein: whey protein from 10:90 to 90:10, more preferably from 20:80 to 80:20, even more preferably from 35:65 to 55:45.

Weight. % protein based on the dry weight of the food composition of the present invention is calculated in accordance with the Kjeldahl method by measuring total nitrogen and using a conversion factor = 6.38 in the case of casein or conversion factor = 6.25 for proteins other than casein. The term “protein” or “protein component” as used in the present invention refers to the sum of proteins, peptides and free amino acids.

The food composition of the present invention preferably contains protein providing from 1.6 to 4.0 g of protein per 100 kcal of food composition, preferably providing from 1.6 to 3.5 g, even more preferably from 1.75 to 2.5 g per 100 kcal of food composition. In one embodiment, the food composition of the present invention contains a protein providing from 1.6 to 2.1 g protein per 100 kcal of the food composition, preferably providing from 1.6 to 2.0 g, more preferably from 1.75 to 2, 1 g, even more preferably from 1.75 to 2.0 g per 100 kcal food composition. In one embodiment, the food composition of the present invention contains protein in an amount of less than 2.0 g per 100 kcal, preferably from 1.6 to 1.9 g, even more preferably 1.75 to 1.85 g per 100 kcal composition. Too low protein per total calories will result in less inappropriate growth and development in infants and young children. When in liquid form, for example, as a ready-to-drink liquid, the food composition preferably contains from 0.5 to 6.0 g, more preferably from 1.0 to 3.0 g, even more preferably from 1.0 to 1, 5 g of protein per 100 ml, most preferably from 1.0 to 1.3 g of protein per 100 ml. Based on dry weight, the food composition of the present invention preferably contains from 5 to 20 weight. % protein, preferably at least 8 weight. % protein, calculated on the dry weight of the entire food composition, more preferably from 8 to 14 weight. %, even more preferably from 8 to 9.5 weight. % protein based on the dry weight of the entire food composition.

To meet the energy needs of infants and young children, the food composition preferably contains from 45 to 200 kcal / 100 ml of liquid. For infants, the food composition contains more preferably 60 to 90 kcal / 100 ml of liquid, even more preferably 65 to 75 kcal / 100 ml of liquid. This calorie density provides the optimal ratio between water intake and calories. For young children who are people of subjects aged 12 to 36 months, the food composition more preferably has a caloric density of from 45 to 65, even more preferably from 50 to 60 kcal / 100 ml. The osmolarity of the composition of the present invention is preferably from 150 to 420 mOsmol / L, more preferably from 260 to 320 mOsmol / L. Low osmolarity is aimed at further reducing the load on the gastrointestinal tract.

When the food composition is in liquid form, a preferred daily volume is in the range of about 80 to 2500 ml, more preferably about 200 to 1200 ml per day. Preferably, the number of feedings per day is from 1 to 10, preferably from 3 to 8. In one embodiment, the food composition is administered daily for a period of at least 2 days, preferably for at least 4 weeks, preferably for a period, of at least 8 weeks, more preferably for a period of at least 12 weeks, in liquid form, the total volume administered daily is from 200 to 1200 ml, and wherein the number of feeds per day is from 1 to 10.

The food composition of the present invention, in liquid form, preferably has a viscosity of from 1 to 60 mPa · s, more preferably from 1 to 20 mPa · s, most preferably from 1 to 6 mPa · s. Low viscosity ensures proper fluid injection, such as proper passage through the entire nipple. Also, this viscosity is very similar to the viscosity of breast milk. In addition, low viscosity leads to normal emptying of the stomach and better energy consumption, which is important for babies who need energy for optimal growth and development. The composition of the present invention is preferably prepared by mixing the powder composition with water. Usually baby formula is prepared in this way. Thus, the present invention also relates to a packaged powder composition, said package provided with instructions for mixing the powder with a suitable amount of liquid, resulting in a liquid composition with a viscosity of 1 to 60 mPa · s. Fluid viscosity is determined using a Physica Rheometer MCR 300 meter (Physica Messtechnik GmbH, Ostfilden, Germany) with a shear rate of 95 s ^-1 at 20 ^o C.

Application

In the context of the present invention, “preventing” a disease or a specific disorder also means “reducing the risk” of a disease or a specific disorder, and also means “treating a person at risk” of developing a specified disease or said specific disorder.

The authors of the present invention found that when consuming the food composition of the present invention, the level of sIgA increases synergistically. This indicates a strengthening of the immune system to protect the mucous membrane.

The effects described here, i.e. an increase in sIgA and / or an enhanced mucosal immune system is observed in a situation comparable to the administration of a food composition not containing a combination of a fermented product and an indigestible oligosaccharide. It was found that these observed effects are also closer to levels observed in breast-fed infants compared to infants fed a standard infant formula.

Surprisingly, the synergistic effect cannot be due to the direct interaction between the indigestible oligosaccharide and lactic acid bacteria in the fermented composition, since the bacteria were inactivated, or the increased activity of intestinal microorganisms, since there was no clear correlation with intestinal pH.

In one embodiment, the food composition of the present invention is used to increase IgA secretion in a human subject aged 0 to 36 months. In one embodiment, the food composition of the present invention is used to increase IgA secretion in a human subject aged 0 to 18 months, even more preferably in an infant of 12 months of age or less, even more preferably in an infant of 0 to 6 months of age. In one embodiment, the food composition of the present invention is used to increase IgA secretion in a young child from 12 to 36 months, most preferably a young child from 18 to 30 months, or 24 months. Preferably, the food composition of the present invention is also used to provide nutrition to said human subject. The food composition of the present invention is preferably administered enterally, more preferably orally.

In one embodiment, the food composition of the present invention is used to enhance the immune defense of a human mucosa in a human subject aged 0 to 36 months. In one embodiment, the food composition of the present invention is used to enhance the immune defense of a human mucosa in a human subject aged 0 to 18 months, even more preferably in an infant of 12 months or less, even more preferably in an infant of 0 to 6 months. In one embodiment, the food composition of the present invention is used to enhance the immune defense of the mucous membrane in a young child from 12 to 36 months, most preferably a young child from 18 to 30 months, or 24 months. Preferably, the food composition of the present invention is also used to provide nutrition to said human subject. The food composition of the present invention is preferably administered enterally, more preferably orally.

In a preferred embodiment, the methods or applications of the present invention are for naturally born infants.

In a preferred embodiment, the methods or applications of the present invention are for full-term babies, preferably for healthy full-term babies.

In this document and in its formula, the verb “contain” (“include”) and its conjugations, used in its non-limiting sense, means that the elements following this word are included, but elements not specifically mentioned are not excluded. In addition, reference to an element using the indefinite article “a” or “an” does not exclude the possibility of more than one element, unless the context clearly requires one and only one of the elements. The indefinite article “a” or “an”, therefore, usually means “at least one”. Weight. % means percentage by weight.

EXAMPLES

Example 1: Synergistic effect of a fermented infant formula and indigestible oligosaccharides on secretory IgA levels in infants

In a randomized, double-blind, controlled, parallel-group, prospective, multi-center, multi-country study, subjects were equally randomized across four treatment groups. In addition, infants exclusively breast-fed from birth (never received infant formula) and their mothers, who intended to continue breast-feeding until the infant was at least 4 months old, were included in the control group for breast-feeding. A total of 350 subjects were registered, of which 280 subjects were randomized to one of the four tested products, and 70 subjects were included in the control group of breastfeeding.

Test group 1: Infant formula 1 is a modified infant formula based on cow's milk for infants who are bottle fed from 0 to 6 months old (Nutrilon 1 sold by Nutricia, Netherlands). The infant formula contains indigestible oligosaccharides (NDO), a mixture of galactooligosaccharides, Vivinal® GOS from FrieslandCampina Domo, with an average degree of polymerization below 6, and fructo-oligosaccharides, RaftilinHP from Orafti, with an average degree of polymerization above 20, in a weight ratio of approximately 9: 1 and in a ratio of about 9: 1 approximately 0.8 g per 100 ml. The milk mixture does not contain a fermented composition.

Test group 2: Infant formula 2 is a modified infant formula based on cow's milk for bottle-fed infants aged 0 to 6 months, and is a fermented infant formula Calisma® (sold by Gallia, France) that includes strains B. breve CNCM 1-2219 and S. thermophilus CNCM 1-1620, which are destroyed by heat treatment after the fermentation process, are bacterial fermentation metabolites such as L (+) - lactate. The amount of L-lactate exceeds 0.05 wt.% Calculated on the dry weight of the composition. NDO is not added. During fermentation, S. thermophilus produces approximately 2 wt.% Based on the dry weight of galactooligosaccharides.

Test group 3: Infant formula 3 is an experimental test mixture and is a modified milk formula based on cow's milk for infants who are bottle-fed, from 0 to 6 months of age. The mixture contains indigestible oligosaccharides (a mixture of galactooligosaccharides, with an average degree of polymerization below 6, and fructooligosaccharides, RaftilinHP from Orafti, with an average degree of polymerization above 20) in an amount of approximately 0.9 g per 100 ml and includes a fermented baby formula Calisma® (sold by Gallia, France), as in IF2 of test group 2. The number of added galactooligosaccharides takes into account the number of galactooligosaccharides present as a result of S. thermophilus .

Control group: Infant formula 4 is a control mixture: a modified infant formula based on cow's milk for infants who are bottle fed, from 0 to 6 months old, not containing indigestible oligosaccharides and not fermented.

All four test milk formulations contained nucleotides and a fat mixture containing long chain fatty acids. The mixtures were similar in caloric content, protein content, fat mixture and contained a similar amount of digestible carbohydrates. The number of live bacteria in test products 2 and 3 was below 10 ³ CFU / g. The mixtures also contained vitamins, minerals, trace elements and other trace elements in accordance with the international directive 2006/141 / EC for infant formula.

At the end of the study, 198 randomized subjects completed the study, while 82 randomized subjects terminated early in the study. There were no statistically significant differences in the number and nature of early termination of participation in the study when comparing the active groups of the tested products with the control group. Subjects were fairly aligned across study groups in terms of demographic and basic characteristics.

For each visit, parents collected a stool sample in stool containers provided by Nutricia Research. Samples were frozen at -20 ^ο C immediately after collection and parents kept at this temperature before transferring samples research staff. In place of the samples stored at -80 ^ο C for subsequent analysis. In each collection, 2 tubes were to be filled halfway. If this was necessary for practical reasons at the study site, other means of transport and storage could be arranged. Fecal secretory IgA levels were determined using standard laboratory methods. Since the distribution of sIgA measurements showed potential asymmetry, a logarithmic transformation was performed for statistical analysis before testing differences in the study groups using ANOVA (analysis of variance). SIgA levels were statistically analyzed based on geometric mean values (effect value = group difference, expressed as the geometric mean ratio due to the logarithmic transformation).

The results are shown in Table 1. For the analysis of the primary data, the analysis according to the prescribed treatment (ITT) was considered the main analysis for the study. After 4 months, when the infants are still fully fed with the infant formula, there is a statistically significant difference (p <0.05) between feces IgA levels in the IF3 group (experimental mixture containing the fermented mixture and indigestible oligosaccharides) and the IF4 control group or IF2 group (Calisma). Compared with the IF1 group (Nutrilon), a trend was observed (p = 0.07). There were no statistically significant differences when comparing IF1 with IF2, IF1 with IF4 or when comparing IF2 with IF4.

Table 1: Concentration of sIgA in feces (μg / g) according to visit - ITT population

SIgA concentration IF1 IF2 IF3 IF4 Breast-feeding Milk mixture Unfermented with NDO Fermented without NDO Fermented with NDO Unfermented without NDO Mean
(standard deviation)
n 714.5 #
(822.8)
42 539.0 *
(639.6)
48 929.4
(889.9)
45 685.3 *
(986.9)
40 1028.0
(603.0)
35 Median 442.6 253.6 742.9 331.4 1051.7 Q1-Q3 142.3-854.1 120.7-756.7 269.0-1517.8 154.5-663.5 489.8-1443.5 Min max 31.6-3634.4 30.2-2345.5 48.4-4994.4 29.2-5356.8 147.3-2329.1

* Compared to test IF3 p <0.05

# Compared to test IF3: p = 0.07

Statistical data are not given in comparison with the control group of breastfeeding, since this control group is not randomized and therefore should not be used in direct comparison with randomized study groups. Statistical analysis for the median has not been conducted, but a similar picture is observed.

The difference in sIgA production between test group 3 and 4 can be represented as the magnitude of the effect, based on geometric mean, which is 1.65. This can be interpreted so that the use of an experimental mixture of IF3 at the age of 4 months leads to an increase of approximately 65% in the concentration of sIgA compared with the use of the control group IF4. A similar value of the effect for the test group 3 in comparison with the test group 1 was 1.53, and for the test group 3 in comparison with the test group 2 was 2.06.

The concentration of sIgA in the group consuming IF3 is synergistically higher than expected based on the results of the group consuming IF1 and 2 and the control IF4, and preferably more resembles the levels observed in the control group of breastfeeding. This indicates a synergistically increased effect on the immune defense of the mucous membrane.

Since the bacteria present in IF3 (and 2) were inactivated by heat treatment, this effect cannot be explained by an increased amount of intestinal lactic acid bacteria originating from the mixture, or by increased fermentation in the intestine of added non-digestible oligosaccharides using these lactic acid bacteria originating from mixtures.

Example 2: Intake of a fermented infant formula with indigestible oligosaccharides increases intestinal sIgA levels

In a randomized, multi-center, double-blind, prospective clinical trial, infants under 28 days of age were enrolled and transferred to receive one of the three mixtures up to the age of 17 weeks:

Test group 1: Infant formula 1 containing 100 ml of 66 kcal, 1.35 g of protein (bovine whey protein / casein in a weight ratio of 1/1), 8.2 g of digestible carbohydrates (of which 5.6 g of lactose and 2 1 g maltodextrin), 3.0 g fat (mainly vegetable fat), 0.8 g non-digestible oligosaccharides, including scGOS (source Vivinal® GOS) and lcFOS (source RaftilinHP®) in a weight ratio of 9: 1. Approximately 50% based on the dry weight of this infant formula, is obtained from Lactofidus ™, a commercially available infant formula sold under the trade name Gallia. Lactofidus ™ is a fermented composition of milk origin and is obtained by fermentation using S. thermophilus , and includes B. breve . Mild heat treatment is used to inactivate lactic acid bacteria. The infant formula contains approximately 0.55% by weight of lactic acid + lactate based on dry weight, of which at least 95% is L (+) - lactic acid / lactate. The composition also contains vitamins, minerals, trace elements and other trace elements in accordance with the international directive 2006/141 / EC for infant formula.

Test group 2: Infant formula 2, similar to infant formula 1, but without indigestible oligosaccharides scGOS and lcFOS.

Test group 3: Infant formula 3, unfermented infant formula with 0.8 g of non-digestible oligosaccharides, including scGOS (source Vivinal® GOS) and lcFOS (source RaftilinHP®) in a weight ratio of 9: 1, and the rest is similar in composition to the infant formula 1.

Stool samples were collected at baseline and 17 weeks after the intervention for physiological and microbiological analysis, similarly described in Example 1. Only samples of a subgroup of subjects with a full set of stool samples (both visits) with enough stool for all analyzes were analyzed. In addition, samples were excluded from infants who used any systemic antibiotics at any time after birth or who used thickeners added to the mixture during the study.

In a selected set of fecal samples, the effect of the infant formula used on secretory immunoglobulin A (sIgA) was evaluated and the results are shown in Table 2.

Table 2. The concentration of sIgA (μg / g) in feces according to the visit

Parameter Statistics provided IF1
(N = 30) IF2
(N = 30) IF3
(N = 30) sIgA (μg / g wet weight of feces) ^W Median
(min-max) 1,424.4 *
(84.9-3346.4) 1245.9
(144.7-3006.1) 499.7 *
(45.6-5145.4) pH ^W Median
(min-max) 6.1 * #
(5.2-7.6) 5,6 #
(5.0-7.3) 6.9 *
(5.6-8.2)

^W Wilcoxon rank sum criteria (W) were used for analysis due to a violation of the assumption of normality and / or the presence of outliers.

* Significantly different values (p <0.05) between groups for the test comparison of interest: IF1 versus IF3.

^# Significantly different values (p <0.05) between groups for the test comparison of interest: IF1 versus IF2.

Again, the concentration of sIgA in the group consuming experimental infant formula 1 is higher than the concentration in the group consuming infant formula 2 or 3, very similar to the results in Example 1. This indicates an increased effect on the immune defense of the mucous membrane.

Interestingly, the effect on fecal sIgA levels did not match the effect on fecal pH. The pH was the lowest in the group consuming the infant formula 2, and intermediate in the group consuming the experimental infant formula 1. The pH reflects the activity of the intestinal microbiota. The low faecal pH of the test groups 1 and 2 is due to the fermentation of the non-digestible oligosaccharides present in the test formula, and the fermentation is the highest in test group 2, while the sIgA levels are the highest in test group 1.

Claims (7)

1. The use of a fermented ingredient and a non-digestible oligosaccharide in the manufacture of a food composition for use in increasing IgA secretion in a human subject from 0 to 36 months old, wherein the food composition contains from 5 to 97.5% by weight of the fermented ingredient based on dry weight, moreover, the fermented ingredient is a milk substrate, which is fermented by Bifidobacterium breve and / or Streptococcus thermophilus, and said milk substrate includes at least one selected from the group consisting of milk, whey, whey protein, whey protein hydrolyzate, casein, casein hydrolyzate and lactose or mixtures thereof, and wherein the food composition contains from 0.02 to 1.5 wt.% of the sum of lactic acid and lactate based on the dry weight of the food composition and contains from 2.5 to 15 wt.% of indigestible oligosaccharide based on dry weight of the food composition, the non-digestible oligosaccharide being oligosaccharide ids with a degree of polymerization (DP) of from 2 to 250, which are not broken down in the intestine by acids or digestive enzymes present in the upper part of the human digestive tract, but which are preferably fermented by the human intestinal microbiota and include galactooligosaccharide and / or fructooligosaccharide. 2. The use according to claim 1, wherein the food composition is a baby formula (milk mixture of the first level), milk mixture of the second level or milk of the third level. 3. The use according to any one of the preceding paragraphs, wherein at least 90% by weight of the total lactic acid and lactate is L (+) - lactic acid and / or L (+) - lactate. 4. The use according to any one of the preceding paragraphs, wherein the food composition comprises Bifidobacterium breve and / or Streptococcus thermophilus. 5. The use according to any one of the preceding paragraphs, wherein the lactic acid bacteria are inactivated to a level below 10 ⁵ , more preferably below 10 ³ CFU / g dry weight of the food composition. 6. The use according to any one of the preceding paragraphs, wherein the food composition comprises protein, a digestible carbohydrate and a lipid, and wherein the protein provides from 1.6 to 4 g per 100 kcal, the digestible carbohydrates provide from 5 to 20 g per 100 kcal, and the lipid provides from 3 to 7 g of lipid per 100 kcal of the food composition. 7. A method of increasing IgA secretion in a human subject from 0 to 36 months of age, comprising administering to the human subject a food composition containing a fermented ingredient and a non-digestible oligosaccharide, wherein the food composition contains from 5 to 97.5% by weight of the fermented ingredient based on dry weight, and said food composition contains from 0.02 to 1.5% by weight of the sum of lactic acid and lactate based on the dry weight of the food composition and contains from 2.5 to 15% by weight of indigestible oligosaccharide based on dry weight food composition, wherein the non-digestible oligosaccharide refers to oligosaccharides with a degree of polymerization (DP) of from 2 to 250, which are not broken down in the intestine by acids or digestive enzymes present in the upper part of the human digestive tract, but which are preferably fermented by human intestinal microbiota and include galactooligosaccharide and / or fructooligosaccharide, and wherein the enzyme the ingredient is a milk substrate that is fermented by Bifidobacterium breve and / or Streptococcus thermophilus, and the milk substrate includes at least one selected from the group consisting of milk, whey, whey protein, whey protein hydrolyzate, casein, casein hydrolyzate and lactose or mixtures thereof.